Iworx, Wayne 2009
Compound Action Potential in the Frog Sciatic Nerve
Purpose: This exercise is designed to familiarize the student with the basic principles of nerve
conduction.
Performance Objectives: At the end of the exercise the student should be able to:
1.
Determine threshold and maximal stimulus.
2.
Measure the conduction velocity of the frog sciatic nerve.
3.
Determine if a nerve is able to conduct electrical signals in two directions.
4.
Demonstrate recruitment using the equipment provided.
5.
Define resting potential, action potential, compound action potential.
6.
Explain what is meant by compound action potential.
7.
Draw a picture of the laboratory setup used for this exercise.
8.
Find and dissect the frog sciatic nerve for placement in a nerve chamber.
9.
Describe the dissection of the frog sciatic nerve
10.
Explain the procedure and significance of a single and double pith of the frog.
Introduction
The interior of a neuron at rest is negatively charged with respect to its exterior. The Resting
Membrane Potential (RMP) is a measure of this difference in charge across a cell’s membrane.
In most organisms, the RMP is between -50 and -80 mV (charge inside with respect to outside)
and is a consequence of negatively charged proteins in the cytoplasm and the membrane’s
permeability to potassium.
Stimulation, like synaptic activity coming from other nerve cells, can transiently reverse the
membrane potential of a neuron (i.e. the interior of the cell goes from negative to positive and
back to negative, again). This event is called an action potential (AP) and takes place in a matter
of milliseconds. At the onset of an action potential, the inside of a cell becomes more positive
due to an increase in sodium permeability. More specifically, when a neuron is stimulated to its
threshold or beyond, voltage gated Na+ channels open, allowing Na+ to enter the cell. As Na+
enters, the interior of the cell becomes more positive (depolarized), particularly in the regions
adjacent to these open channels. Since depolarization acts as a stimulus to open more v-gated
Na+ channels, new action potentials are generated along the length of the axon. Na+ channels do
not remain open indefinitely, however, and the cell never reaches the equilibrium potential of
sodium. This is due to the fact that voltage-gated sodium channels close shortly after they open
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Iworx, Wayne 2009
and voltage-sensitive potassium channels open. With Na+ channels closed and K+ channels
open, potassium ions exit the cell and cause the membrane to repolarize (hyperpolarize) towards
its resting level. Voltage-gated potassium channels close and then sodium channels are ready to
open once more.
In this laboratory, you will record action potentials from the Sciatic Nerve of a frog. The Sciatic
Nerve is composed of thousands of individual axons grouped together and enclosed in a
connective sheath. The nerve contains sensory, motor and autonomic axons that individually
vary in diameter, myelination, threshold and speed of conduction. Large, myelinated axons with
the fastest conduction velocities are known as Type A fibers. Type B fibers are also myelinated,
but have smaller diameters and slower conduction velocities. Type C fibers are very small,
unmyelinated axons. When a large stimulus is delivered to the nerve, many axons in the nerve
respond and the recorded potential is the summation of these axons firing simultaneously. This
potential is known as the compound action potential (CAP).
Equipment Required
Laptop computer, iWorx/214 orr 204 and USB cable
Nerve Chamber
AAMI cable and nerve chamber leads (red and black)
Glass hooks
Stimulator cable
Grounding adapter or cable
Frog Ringer's solution at two temperatures:
25ml per station chilled on ice
100 ml per station at room temperature
Start the Software
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1.
Click the Windows Start menu, move the cursor to Programs and then to the
iWorx folder and select LabScribe; or click on the LabScribe icon on the Desktop
2.
When the program opens, select Load Group from the Settings menu.
3.
When the dialog box appears, select IP ------ and then click Load.
4.
Click on the Settings menu again and select Animal Nerve, Compound Action
Potential.
.
5.
After a short time, LabScribe will appear on the computer screen as configured by
Compound Action Potential setting in Scope mode.
6.
Press the mode button on the Channel 1&2 section of the iWorx box until the
EMG light is illuminated.
7.
The equipment is ready. Refer to the picture at the end of the lab if the cables are
not connected.
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The Dissection
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1.
Review the movie provided by the instructor.
2.
To begin dissection, retrieve a frog from your instructor and place it in a
dissecting tray.
3.
Remove the skin from the legs by making an incision through the skin and around
the entire lower abdomen. Cut the connections between the skin and the body-especially around the base of the pelvic girdle.
4.
Use stout forceps to pull the skin off the frog in one piece (like a pair of pants).
5.
Place the frog with its dorsal side up. Moisten the exposed tissue (legs) with
Ringer's solution and place a wet paper towel (saturated with Ringers solution)
over one of the legs of the frog so that it is completely covered and wet.
6.
Use forceps to separate the muscles of the thigh (the leg not covered with the
paper towel). The muscles are surrounded by connective tissue called fascia, and
the large medial and lateral muscles on the dorsal side of the upper leg are joined
to each other by a fusion of their fascia along a thin "white line". Grab the muscle
groups on either side of the "white line" with forceps, and firmly pull the muscle
groups apart. The fascia will tear.
7.
Pin the muscles apart so that more underlying muscle is visible. This should also
expose the cream-colored Sciatic nerve lying deeply between the muscles. The
Sciatic nerve is covered with fascia, which also includes some blood vessels.
8.
Use a glass hook to separate the nerve from the fascia and the vessels. If possible,
avoid cutting the blood vessels. If bleeding does occur, rinse away the blood with
lots of Ringer's solution. Free the nerve from the knee joint to the pelvis.
9.
Use the glass hook to place a suture thread under the nerve. Move the thread as
close to the knee joint as possible. Ligate (tie off) the nerve; you may observe calf
muscle fibrillation or foot movement as the knot is tied off. Be sure the knot is
tied tightly. Cut the nerve between the knot and the knee joint. Keep the exposed
nerve moist at all times with Ringer's solution.
10.
Carefully separate the muscles of the pelvis to expose the Sciatic nerve.
Remember to rinse any blood away with Ringer's solution. The Sciatic nerve
enters the abdomen of the frog through an opening at the end of the urostyle, a
bone that forms part of the pelvis.
11.
Carefully expose the remainder of the nerve through an opening along the lateral
side of the urostyle. To avoid cutting the nerve, lift the end of the urostyle with
forceps as you cut the muscle away from the urostyle with blunt scissors. Cut
along the urostyle from its tip to the vertebral column.
12.
Deflect the muscle away from the urostyle to expose the Sciatic nerve. Use a glass
hook to separate connective tissue from the nerve and to place a piece of suture
thread under the nerve. Move the thread as high as possible on the nerve to obtain
as large a section as possible. Ligate (tie off) the nerve; the leg may jump again as
the knot is tied tightly.
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Iworx, Wayne 2009
13.
Cut the nerve between the knot and the vertebral column. Keep the exposed nerve
moist at all times.
14.
Use forceps to grasp the suture thread at the proximal end (end closest to head)
and lift the nerve out of the body cavity. Do not pinch or stretch the nerve.
Remove any connective tissues, blood vessels, or nerve branches that may still
keep the nerve attached to the frog. Continue to grasp the suture to lift the nerve
until it is clear of the abdomen, the pelvis, and the thigh.
15.
Grasp the suture at either end to remove the nerve from the body entirely. Place
the nerve across the gold-colored electrode pins in the nerve bath. Add a small
quantity of Cold Frog Ringers to the bottom of the chamber. The Frog Ringers
should not touch the gold-plated electrode pins.
16.
Cover the chamber with a glass slide.
The Nerve Chamber
1.
The proximal end of the nerve (end connected to the spinal column) should be over the stimulating electrodes, and the
distal end (from knee region) should be over the recording electrodes.
2.
The ligature (knot) on the distal end should be located between the two recording electrodes in the nerve bath. Make
sure that the nerve is in contact with the stimulus and recording electrodes.
3.
Place a few drops of frog ringer's solution into the chamber. The level of the frog ringers must not contact the
electrodes. Cover the chamber with glass microscope slides. The picture below show the nerve chamber and the
proper connectionof the leads.
Exercise 1: The Compound Action Potential
Goal: To apply a brief stimulus at the proximal end of the nerve and record a compound action
potential from the distal end.
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Procedure
1.
Select <stimulator> from the menu bar. .
The stimulus amplitude should be 0.25 V and the pulse width should be 0.1ms.
1
2.
Click Record to stimulate and record from the nerve. LabScribe is set to use
Scope mode and to display Repetitive sweeps. This means that the nerve will be
stimulated again, after the preceding sweep is completed, A new recording of the
nerve response replaces the previous sweep on the Main window. Scope will
continue to stimulate the nerve and display new compound action potentials until
the Stop button is clicked. Click the Stop button to preserve the latest sweep
displayed in the window.
3.
A mark line appears on the left side of the screen to indicate the point in time
when the stimulus was delivered to the nerve. There may be a stimulus artifact at
the mark (the artifact may appear as a square-shaped wave). The compound
action potential usually reaches a peak a few milliseconds after the artifact.
4.
If the tracing is upside down, right click and select invert.
6.
If the tracing looks like the one below, press print screen to preserve your results.
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Iworx, Wayne 2009
: The compound action potential.
Exercise 2: Stimulus and Response
Goal: To quantify the relationship between stimulus intensity and response of the nerve.
Procedure
1.
Begin a new file by selecting New from the File menu.
2.
Use the arrow buttons in the stimulator panel to change the stimulus amplitude to
0.00 V (zero). Click the Apply1 button to the right of the stimulator panel to
effect the change in the stimulus.
3.
Click Record to stimulate the nerve with 0.00V. A flat line should be observed.
Click Stop to display the sweep in the Main window.
Optional1- Click the 1-Cursor icon in the LabScribe toolbar, type"0.00V" on the
comment line to the right of the Marks button. Press the Enter key on the
keyboard to attach the comment to the sweep.
4.
: The LabScribe toolbar
6.
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Change the stimulus to 0.05 Volts. Click Record. . After the trace appears, click
Stop. On the recording window, there may be a small peak at the stimulus mark;
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Iworx, Wayne 2009
this is the stimulus artifact. You may also observe a deflection in the trace to the
right of the stimulus artifact. This deflection represents the response from one
or more neurons in the nerve. Type "0.05 V" on the comment line, and press the
Enter key to attach the comment to the sweep.
7.
Click the 2-cursor icon in the LabScribe toolbar and determine the amplitude of
the action potential. Record your data
8.
Continue to increase the stimulus in 0..05 V increments until no further increase
in response amplitude is observed. This is the maximum response. Record the
results on the table on your data sheet. Take the measurements after each
stimulus.
Data Analysis
Plot the data on a piece of graph paper or use Excel with the CAP voltage on the y-axis and the stimulus voltage
on the x-axis. Determine the maximal stimulus using this graph.
Questions
1.
Can action potentials from a single neuron vary in amplitude? Why or Why not?
Predict and describe how a single neuron would respond to changes in stimulus
amplitude (like exercise 2 above).
____________________________________________________________________
____________________________________________________________________
_____________________________________________________________________
2.
Were there stimuli that failed to elicit a response from the nerve? Explain why.
____________________________________________________________________
____________________________________________________________________
3.
At what voltage did you observe the first CAP?
_____________________________________________________________________
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Iworx, Wayne 2009
4.
Why does a CAP increase in amplitude as stimulus amplitude is increased? What
does the maximum response represent? Explain.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
Exercise 3: Conduction Velocity
Goal: To measure the velocity of action potential conduction
Procedure
1
1.
Change the stimulus amplitude (Amp) to the lowest voltage
that creates a maximal compound action potential. Click the
Apply button to finalize the change in the stimulus
amplitude
2.
If necessary, lower the level of Ringer’s solution in the nerve
bath chamber below the nerve. Remove the solution with a disposable pipet.
3.
Connect the black (-1) recording lead wire to the first recording electrode that is
next to the ground electrode. Make sure the red (+1) recording lead wire is on the
outermost recording electrode.
4.
Click Record to stimulate the nerve. Type Short Path in the Mark box to the right
of the Mark button. Press the Enter key on the keyboard to attach this notation to
the recording.
5.
Move the black (-1) recording lead wire to another recording electrode that is
farther from the ground electrode. Do not move the red (+1) recording lead wire.
6.
Click Record to stimulate the nerve. Type Long Path in the Mark box to the right
of the Mark button. Press the Enter key on the keyboard to attach this notation to
the recording.
Since there can be problems with the software and other issues, collect your data as you go.
Periodically save your data to the desktop.
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Iworx, Wayne 2009
7.
Measure the distance between the two positions of the negative (-1) recording
electrode.
8.
Select Save in the File menu.
Data Analysis
1.
Use the Sweep Selection bar at the bottom of the Main window to display the
first sweep needed to calculate the conduction velocity ofthe nerve. Click on the
tab of that sweep and it will appear on the Main window.
2.
Transfer the sweep to the Analysis window using the Analysis window icon in
the toolbar or the Analysis listing on the Windows menu.
3.
Display the second sweep needed to calculate the conduction velocity by clicking
on its tab in the Sweep. Selection bar at the bottom of the Analysis window.
4.
Measure the time between the peaks of the two compound action potentials
displayed on the Analysis Window
5.
Record the T2-T1 value in your notebook.
6.
Calculate the conduction velocity (in m/s). For example: If T2-T1 = 0.5ms and
distance 10mm
Velocity = (distance stimulus wires moved)/ 0.5 ms = 10mm/0.5ms = 20mm/ms
= 20m/s
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Iworx, Wayne 2009
7.
Record this value on your data sheet.
Exercise 4: Bidirectionality
Goal: To examine whether an action potential travels in one or both directions and at what
velocity.
Procedure (Check with instructor first)
1.
Return the electrodes to their original positions.
2.
Reverse the position of the electrodes attached to the pins on the nerve bath. Place
the stimulating electrodes on the distal end of the nerve, where the recording
electrodes used to be, and vice versa.
4.
Stimulate the nerve with the same amplitude used in the last exercise.
5.
Measure the conduction velocity of the nerve as done in the previous exercise.
Questions
1.
Do you record a CAP from the proximal end of the nerve (when you stimulate the
nerve from the distal end)? If yes, explain why you believe the nerve is
conducting in both directions.
______________________________________________________________________
______________________________________________________________________
2.
What is the conduction velocity for the nerve when stimulated in the reverse
direction? Is it similar to the value recorded when the nerve was stimulated from
the proximal end? Why or why not?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
1
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Periodically save your data to the desktop.
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Iworx, Wayne 2009
Exercise 4: Effect of anesthetic on the Compound Action Potential
Goal: To observe the effect of ether on the compound action potential
1. Apply a maximal stimulus to the nerve to insure that everything is working properly.
2. Using a disposable pipet apply a small amount of anesthetic along the nerve. Be careful
not to touch the nerve with the pipet.
3. Apply maximal stimuli every 5 seconds for 30 seconds. Record you results.
Cleanup and Disposal
Wrap the frog in paper towels and discard in the trash can.
Clean all dissecting equipment with soap and water. Blot them dry with paper towels and
return to the tray.
Disconnect the nerve chamber , pour off the frog ringers and rinse with distilled water.
Place the cleaned nerve chamber on a paper towel at your station.
Turn off the iWorx program and shut down the computer.
Spray your work area with disinfectant before leaving the laboratory.
Neurophysiology of Nerve Impulses
Data Sheet
Activity: Visualizing the Compound Action Potential:
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Stimulus Voltage versus amplitude of compound action potential (CAP)
Stimulus
Voltage
CAP
voltage
Stimulus
Voltage
CAP
voltage
0
.05
.10
.15
.90
.95
1.0
1.05
.30 .35
1.10
.40 .45
1.15
1.20
.50 .55
1.25
.60
1.30
.65
1.35
.70
.75 .80
1.45
1.50
.85
A. Relation between stimulus Intensity and Compound Action Potential and Maximal
Stimulus
Stimulus Threshold:
______________
Maximal Stimulus Voltage:
______________
Maximal CAP:
______________
B. The Velocity of a Nerve Impulse:
T2-T1:____________
mm between electrodes:__________
Velocity of a nerve impulse (show your work):
How does this compare with values given in lecture or text?
C. Bidirectionality of a nerve.
T2-T1:____________
mm between electrodes:__________
Velocity of a nerve impulse (show your work):
1
Since there can be problems with the software and other issues, collect your data as you go.
Periodically save your data to the desktop.
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Iworx, Wayne 2009
1
Since there can be problems with the software and other issues, collect your data as you go.
Periodically save your data to the desktop.
13